1. Field of the Invention
This invention relates generally to the field of chemistry, and in particular is directed to a new method of detecting in situ immunohistochemical epitopes and nucleic acid sequences using an enzyme-mediated reaction for the localized deposition of metal atoms.
2. Description of Related Art
Tissue staining is an ancient art by modern standards that goes back over one hundred years. Recently, efforts have been made to automate the procedure of applying different types of chemical and biochemical stains to tissue sections. Instruments that have been invented for this purpose include the Ventana Medical Instruments' line of dual carousel-based instruments such as the 320, ES®, NexES®, BENCHMARK®, and the BENCHMARK® XT. Patents that describe these systems include U.S. Pat. Nos. 5,595,707, 5,654,199, 6,093,574, and 6,296,809, all of which are incorporated herein by reference in their entirety. Another type of automated stainer is the TechMate® line of stainers, described in U.S. Pat. Nos. 5,355,439 and 5,737,499, both of which are incorporated herein by reference in their entireties.
Various manual detection chemistries have been developed for histochemistry over the years. Generally, once a molecular marker or target of interest has been identified through biomolecular studies it needs to be rendered visible under the light microscope for a Pathologist or other medical specialist to interpret. The first detection step involves an anti-target primary antibody detectably labeled with biotin, digoxigenin, fluoroscein or other hapten being used to locate the biological target of interest. Next, an anti-hapten secondary antibody conjugated to an enzyme or other reporter molecule is used to locate the primary antibody. Typical enzyme systems are known to those of ordinary skill and include horseradish peroxidase or alkaline phosphatase. These enzymes then catalyze the precipitation of a chromogenic substrate in the immediate vicinity of the primary-secondary antibody complex. Chromogens such as nitro blue tetrazolium (NBT/BCIP); 3,3′-diaminobenzidene tetrahydrochloride (DAB); and 3-amino-9-ethylcarbazole (AEC) are well-known. Alternately, enzyme substate interactions may produce chemiluminesent signals, which can be captured on a photographic film.
Other labels include: 125I-labeling of the secondary antibody, which can be detected using a photographic film; fluorescein isothiocyanate-labeled second antibody, which can be detected using UV light; 125I-labeled Protein A, which can be used instead of a secondary antibody, as it will bind to the Fc region of IgG molecules; Gold-labeled secondary antibody, which is directly visible as a red color when they are bound with the secondary antibody to the primary antibody; Biotinylated secondary antibody, which when incubated with the secondary antibody, then incubated with enzyme-conjugated avidin which binds strongly to the biotin, will give an enhanced signal, as multiple biotin molecules can be attached to a single antibody molecule. Enzymes typically used include alkaline phosphatase (“AP”) or horseradish peroxidase (“HRP”).
Metallic enhancement of immunohistochemical detection is taught in U.S. Pat. No. 5,116,734 (Higgs et al.), incorporated herein by reference in its entirety. The '734 patent is directed to a composition of matter and a process for detecting the presence of an oxidative catalyst in a biological sample. The composition comprises a precipitate formed by oxidation of a chromogenic substrate in the presence of the catalyst, together with two or more co-precipitated reduced metals. A strong signal is formed with which to detect an oxidation catalyst which is localized to a target molecule. Target molecules may be nucleic acids, antibodies or cell surface antigens. In particular, Higgs et al. rely on a chromogenic precipitate and two or more metals, for the purpose of detecting an oxidative catalyst. Merchanthaler et al., J. Histoch. And Cytochem., 37:10 1563-65 (1989) teach silver intensification of the oxidatively polymerized DAB by pre-treating the DAB with nickel ions.
A more recent example of metallic enhancement of immunohistochemical detection includes U.S. Pat. No. 6,670,113 (Hainfeld). The '113 patent is directed to a method of producing metal in a zero oxidation state from metal ions, comprising: providing metal ions of at least one metal selected from cesium, periodic table group 1b, 2a, 4a and 8, an oxygen containing oxidizing agent and a reducing agent selected from at least one of hydroquinone, a hydroquinone derivative or n-propyl gallate; providing an oxido-reductase enzyme; combining the enzyme with the metal ions, oxidizing agent and reducing agent; and reducing at least some of the metal ions to metal in a zero oxidation state. In particular, silver ion reduction to silver metal in proximity to horseradish peroxidase when exposed to hydrogen peroxide and hydroquinone is taught.
There continues to be a need for better biochemical techniques for visually identifying immunohistochemical epitopes and DNA targets of interest via bright field light microscopy.